Analysis of Cleavage Activity of Dengue Virus Protease by Co-transfections

The genome of the dengue virus codes for a single polypeptide that yields three structural and seven non-structural (NS) proteins upon post-translational modifications. Among them, NS protein-3 (NS3) possesses protease activity, involved in the processing of the self-polypeptide and in the cleavage of host proteins. Identification and analysis of such host proteins as substrates of this protease facilitate the development of specific drugs. In vitro cleavage analysis has been applied, which requires homogeneously purified components. However, the expression and purification of both S3 and erythroid differentiation regulatory factor 1 (EDRF1) are difficult and unsuccessful on many occasions. EDRF1 was identified as an interacting protein of dengue virus protease (NS3). The amino acid sequence analysis indicates the presence of NS3 cleavage sites in this protein. As EDRF1 is a high-molecular-weight (~138 kDa) protein, it is difficult to express and purify the complete protein. In this protocol, we clone the domain of the EDRF1 protein (C-terminal end) containing the cleavage site and the NS3 into two different eukaryotic expression vectors containing different tags. These recombinant vectors are co-transfected into mammalian cells. The cell lysate is subjected to SDS-PAGE followed by western blotting with anti-tag antibodies. Data suggest the disappearance of the EDRF1 band in the lane co-transfected along with NS3 protease but present in the lane transfected with only EDRF1, suggesting EDRF1 as a novel substrate of NS3 protease. This protocol is useful in identifying the substrates of viral-encoded proteases using ex vivo conditions. Further, this protocol can be used to screen anti-protease molecules. Key features • This protocol requires the cloning of protease and substrate into two different eukaryotic expression vectors with different tags. • Involves the transfection and co-transfection of both the above recombinant vectors individually and together. • Involves western blotting of the same PVDF membrane containing total proteins of the cell lysate with two different antibodies. • Does not require purified proteins for the analysis of cleavage of any suspected substrate by the protease.

°C up to one month.Note: From this 10× stock, 500 mL of 1× PBS solution can be prepared: mix 50 mL of 10× PBS with 450 mL of H2O (can be stored at 25 °C for a week).Use double-distilled water (ddH2O) (autoclaved) for preparing 1× PBS solution.

Blocking buffer
Reagent Final concentration Quantity BSA/skimmed milk powder PBST 2% or 7% 1× 0.2 or 0.7 g 10 mL Note: Prepare fresh when required.7% blocking buffer solution is required for the blocking the PVDF membrane.Optional: 2% blocking buffer solution is required for preparing antibody dilutions.
5. Add Mix-1 to Mix-2 and incubate for 20 min to form a plasmid-Lipofectamine complex.6. Add the complex by gentle pipetting onto the cells drop by drop and further incubate the cells at 37 °C in a CO2 incubator for 5-6 h. 7. Replace the media with fresh complete DMEM and allow the cells to grow for 48 h. 8.After 48 h of transfection, remove the media from the plates and add 1 mL of 1× PBS.
a. Scrap the cells with a cell scrapper and collect the cells by pipetting into a fresh 1.5 mL Eppendorf tube.b.Centrifuge at 800× g for 5 min.c.Wash the cells with 1 mL of 1× PBS and centrifuge at 800× g for 5 min.d.Repeat the wash step twice if pelleted cells still contain traces of DMEM medium.9. Add 200 μL of RIPA buffer and 20 μL of 1× Protease inhibitor cocktail to the cell pellet and resuspend gently.a. Vortex the suspension thrice at 5 min intervals and keep on ice for 30 min.b.Centrifuge the cell suspension at 20,000× g for 15 min.c.Collect the supernatant into a fresh Eppendorf tube as whole-cell lysate.10.Quantify the total protein of the lysates by Bradford reagent.
a. Resolve 60 μg of the quantified protein on 10% SDS PAGE.
Optional: Before proceeding with the cell harvesting step C8, GFP expression can be analyzed using a fluorescence microscope.Note: GFP expression was analyzed to confirm the transfection.

D. Western blotting
1. Transfer the above resolved proteins onto PVDF membrane at 80 V current for 3 h (or 50 V overnight) using a western blot transfer unit in 1× transfer buffer.

E. Stripping and re-probing the PVDF membrane
Stripping is performed to re-probe the membrane with the different antibodies.All the steps for stripping can be carried out at room temperature.1.From the above step D9, use the membrane for stripping.a. Wash the membrane with ddH2O two times for 5 min each on the rocker with medium agitation.b.Repeat the wash step two times with 1× PBST for 5 min each on the rocker with medium agitation.c.Add ~10 mL of stripping buffer and incubate the membrane for 30 min on the rocker with medium agitation at room temperature.d.Discard the stripping buffer and wash the membrane with ddH2O for 5 min on a rocker.e. Wash the membrane with 1× PBS two times for 5 min each on the rocker.f.Repeat the wash steps with 1× PBST two times for 10 min each on the rocker.g.The membrane is ready for the re-probing with different antibodies.2. For re-probing with the different antibodies, begin with the blocking steps as mentioned in the above western blotting steps (D4-D9).

Data analysis
In this protocol, we analyzed EDRF1 cleavage by co-transfection followed by western blotting.The western blot data was obtained using anti-Myc tag antibody for pCDNA 3.1 c-myc EDRF1 and GFP-tag antibody for pEGFPN1 NS2BNS3pro expressions.In pcDNA3.1 c-myc vector containing EDRF1 as an insert, the EDRF1 band was detected as intact showing the presence of expressed EDRF1 alone (Figure 2, lane 2).As expected, no expression was observed in pcDNA3.1 c-myc vector alone.Importantly, the expressed EDRF1 completely disappeared in the presence of protease in co-transfected pCDNA3.1 c-myc EDRF1 and pEGFP-N1 NS2BNS3pro conditions, suggesting EDRF1 as a substrate of protease (Figure 2, lane 4).pEGFP-N1 vector alone and pEGFP-N1 NS2BNS3pro lysates were loaded as controls (Figure 2, lanes 11 and 12).To confirm the expression of NS2BNS3pro in the above experiment, we have checked the expression of protease using anti-GFP antibody (pEGFPN1-NS2BNS3pro) after stripping the same membrane.It was observed that, in co-transfected vectors and pEGFP-N1 vector alone, GFP was expressed.In pcDNA3.1 c-myc EDRF1 and pEGFP-N1 NS2BNS3pro cotransfected cell lysates, the protease was detected, thus confirming the expression of NS2BNS3pro in the cotransfected conditions (Figure 2, lane 10).pEGFP-N1 NS2BNS3pro was also found to be expressed alone, which is a control (Table 2).We expect that this analysis will be useful in identifying the novel substrates of viral encoded proteases, as this protocol is simple, easy, and quick to perform.Further, this protocol can be used to evaluate antiprotease molecules.

Validation of protocol
This protocol was described in our published article in iScience (2023), https://doi.org/10.1016/j.isci.2023.107024.We have repeated the protocol and found that it is easy to execute, and the outcome is consistent.In vitro pulldown assay followed by mass spectrometry identification/western blotting confirmed the interaction between EDRF1 and protease.In vitro sequence analysis suggested the existence of a total of five protease cleavage sites in EDRF1.Further, superimposed models of EDRF1 and protease indicated the location of the cleavage site within the catalytic triad of protease.The above observations support the outcome of the protocol described.

General notes
This protocol requires either the two differently expressing vectors with different tags or proteins specific for analyzing the expression, if using the same tag for the co-transfected plasmids.The protocol is more convenient to be performed with two different tagged vectors.Proper use of sterile equipment and reagents in cell culture will allow maintenance of the aseptic environment and minimize contamination.Collect the cells from the certified cell repositories (NCCS, Pune, India, or ATTC).Culture and split the cells one at a time or perform the splitting of cells on alternate days.Prepare the cryomedium fresh, if possible.Use cell culture grade DMSO or glycerol or commercially available cryomedium.Check the expiry date of the FBS, media, and antibiotics used for culturing the cells.

Cell culture media color changes
Highly confluent dishes.CO2 levels are low.Bacterial contamination.
Thaw a new cryovial or split the cells as needed.Set the CO2 levels to 5% and maintain humidity.Add antibiotics and wash the cells with 1× PBS (cell culture grade).Discard the media and sterilize the laminar hood cabinets and CO2 incubator.Use 5%-7% DMSO in cryomedium.Use 90% FBS during cryofreezing.Prepare cryovials with 80% confluent freshly split cells.

Cell growth slows
Less or inaccurate supplemental cell culture components.Cell culture conditions (temperature, humidity, and CO2 levels).Protein of interest not expressing.
Begin with fresh cryovials.Growth media with 10%-12% FBS will be good for attaining a good confluency.Keep the incubator at 37 °C with 5% CO2 and water tray.Grow the cells without antibiotics.

Co-transfection not working
Transfection reagents may not be suitable.Cell lines may not be efficient for cotransfection.Co-transfecting plasmids not expressing together.
Check the quantity and quality of plasmids.Use high quality plasmid isolation kits (Thermo MidiPrep or High Pure Links Kits) Lipofectamine 2000 works best for most cell lines.Lipofectamine 3000 can also be used.Check the transfection efficiency before proceeding with the co-transfection experiments.Perform and analyze the transfection of the individual plasmids that need to be co-transfected.

Western blot of the co-transfected plasmids
Antibody not detecting the proteins in co-transfecting plasmids.No signal in the blot.High background.
Optimize the transfection timing for the co-transfected plasmids.Forty-eight hours of co-transfection give conclusive results.Use individual plasmids as controls to confirm the transfection.Optimize the antibody dilution (1:500 to 1:3,000).Use specific tagged antibodies or protein-specific antibodies.Use species-specific secondary antibodies (anti-mouse or anti-rabbit HRP conjugated).Optimize the primary and secondary antibody incubation times and washing steps.Optimize the blocking buffer (5%-7% BSA or skimmed milk).Use 1× PBST and up to 0.3% Tween-20.

5 Published
Cite as: Gandhi, L. and Venkataramana, M. (2024).Analysis of Cleavage Activity of Dengue Virus Protease by Cotransfections.Bio-protocol 14(5): e4946.DOI: 10.21769/BioProtoc.4946.Note: From this 10× stock, 1× transfer buffer can be prepared: dissolve 100 mL of 10× transfer buffer in 200 mL of methanol and make up the volume with water up to 1 L. Store at 4 °C; this can be reused for two weeks.

2 .
Stain the PVDF membrane with Ponceau S stain.a. Prepare 50 mL of fresh Ponceau S stain solution.Published: Mar 05, 2023 b.Add 4-5 mL of Ponceau S stain onto the PVDF membrane and immerse the membrane completely.c.Incubate the membrane for 5 min with slow agitation on the rocker.d.Remove the Ponceau S stain and collect it in an Eppendorf tube for reuse (at least twice).e. Add ddH2O water to wash out the excess stain and, as the bands start appearing immediately, record the image.3. Wash the membrane with 1× PBST until the Ponceau is removed completely.Note: Ponceau stain must be removed as it may hinder the blocking step.4. Prepare 7% blocking buffer and block the membrane for 2 h at room temperature under gentle shaking.Note: Blocking step needs to be optimized for each antibody.7% Blocking buffer is used to avoid nonspecificity.5. Rinse the membrane once with 1× PBST and add the primary antibody solution [anti-Myc Tag (1.4 μg/mL, 1:1000) in 1× PBST or 2% blocking buffer solution].Note: 2% Blocking buffer is used for diluting the primary antibodies.a. Incubate the membrane at room temperature for 2 h or at 4 °C overnight with gentle rocking.Note: Incubation time and dilution of antibody need to be optimized based on the non-specific bands.6. Wash the membrane with 1× PBST three times for 10 min each on a rocker with mild agitation.7. Add secondary antibody (anti-mouse IgG HRP conjugated; 1:10,000) diluted in 1× PBST.8. Incubate the membrane for 2 h at room temperature.9. Wash the membrane with 1× PBST three times for 15 min each with high agitation.10.Develop the washed membrane with western blot Femto LUCENT™ PLUS-HRP substrate solution and capture the image using Chemidoc Image System (Bio-Rad) (see Troubleshooting 6).

11 Published: Mar 05, 2023 Figure 2 .
Figure 2. Western blotting analysis of co-transfected lysates.A. Ponceau image after electroblot transfer.The lysate loaded is indicated on each lane.B. and C. Simulated images of western blotting probed with anti-Myc and anti-GFP antibodies.

3 .
Cells did not attach after being obtained from cryogenic stage Cryomedium shows toxicity.Too many apoptotic cells.Less cell numbers during cryofreezing.Higher number of continuous passages.
Note: Adjust pH to 7.4.Stock can be stored at